Iron is an important nutrient, required in almost every aspect of cellular function. However, at physiological pH and under oxidizing condition, it is not very soluble. How living organisms sequester iron for cellular utilization is therefore a fundamental question of vital importance. Also, in the presence Of 02, free Fe2+ ions are extremely toxic, capable of generating hydrogen peroxide, superoxide, and other reactive oxygen species that can attack and destroy important cellular molecules. Ferritin is unique in the sense that it performs dual functions of iron detoxification, by oxidizing the Fe2+ ions in solution, and iron sequestration, by storing the oxidized Fe3+ ions in its inner protein cavity in the form of ferrihydrite mineral. However, despite the importance of ferritin functions and decades of research efforts, the mechanism by which ferritin catalyzes the Fe2+ oxidation (ferroxidation) and directs the oxidized products to form the mineral core (mineralization) is still poorly understood. This is partly due to the complexity of the ferritin molecule and partly due to the fact that the methods used in previous studies were either indirect or lacked the required spectroscopic resolution to monitor the complex reaction catalyzed by ferritin. In this application, we propose to employ M[unreadable]ssbauer spectroscopy in conjunction with the rapid freeze-rapid quench kinetic technique to investigate the mechanism of ferritin ferroxidation and mineralization. Three different recombinant ferritins, the frog H and M ferritins, and the E. coli bacterioferritin, are to be examined. Results obtained from our preliminary studies demonstrate that this combined kinetic/spectroscopic approach provides the necessary time resolution for obtaining kinetic information and the required spectroscopic resolution for distinguishing, quantifying and characterizing the multiple Fe species generated during the ferroxidation and mineralization processes. Other complementary spectroscopies, such as EPR, ENDOR, EXAFS, and resonance Raman will also be employed to obtain further structural information on these reaction intermediates. Site-specific mutants will be engineered, produced and subjected to kinetic/spectroscopic investigations for the purpose of defining the ferroxidase site, the Fe transport pathways, and the functional roles of certain key residues. A series of double-mixing rapid freeze-quench Mossbauer investigations using 57Fe and 56Fe isotopes are particularly designed to address questions concerning the dynamics of the ferritin function. Detailed mechanistic insights into the processes involved in ferritin ferroxidase reaction and mineral core formation are expected to emerge from these proposed studies.